American Journal of Pathology, Vol. 140, No. 3, March 1992 Copyrght C American Association of Pathologists
Rapid Communication Dual Role of Tumor Necrosis Factor-a in Angiogenesis Luis F. Fajardo,* Helen H. Kwan,* Joe Kowalski,t Stavros D. Prionas,t and Anthony C. Allisont From the Departments of Pathology' and Radiation Oncology, Stanford University School ofMedicine, and the Veterans Affairs Medical Center, Palo Alto, California, and the Institute ofBiological Science,t Syntex Research,
Palo Alto, California
The role of tumor necrosis factor-ot (TNF; cachectin) in angiogenesis has been controversial. In vitro TNF inhibits proliferation of endothelial cells (EC) whereas in the cornea it appears to stimulate vessel growth. The authors tested TNF in their recently developed disc angiogenesis system (DAS), designed to measure the proliferation of microvessels. The DAS, implanted subcutaneously in mice, was either fitted with a central pellet containing mouse recombinant TAF (mrTNF), or exposed to mrTNF delivered subcutaneously by an osmotic minipump. Low doses of mrTNF (0. 01-1 ng) induced angiogenesis, which was maximum at 0.1 ng whereas high doses (1, and 5 jig) inhibited it. Subcutaneous mrTNF delivered at the (high) rate of 15-60 ng/hr for 14 days inhibited angiogenesis. These results indicate bimodal; dosedependent opposing effects and explain some of the in vitro versus in vivo paradoxical results. TAF (native or exogenous) may have opposing effects on micovessels of neoplasms and inflammatory reactions, depending on its local tissue concentrations. (Am J Pathol 1992, 140:539-544)
Angiogenesis, the proliferation of microvessels in the developed vertebrate, is a process particularly important in neoplasia, inflammation, and wound repair.1'2 Some cell products2'3 and stromal components 4'5 that regulate it have been identified. Among the substances that may influence angiogenesis is tumor necrosis factor-a (TNF), a cytokine mainly produced by macrophages. TNF has
multiple well-recognized biological activities.6'9 However, its function in angiogenesis is incompletely understood because of discrepant observations. Several in vitro studies indicate that TNF inhibits the proliferation of cultured capillary endothelial cells (EC), 1l12 or microvascular sprouts,13 and is cytotoxic for stimulated EC.1" Those effects suggest that in vivo TNF should inhibit angiogenesis. However, two studies reported that in the cornea of rabbits and rats, human and mouse TNF, respectively, induced angiogenesis.12,14 To clarify this paradox, we have tested TNF in a new in vivo system (developed in our laboratory15) which allows easy quantification of vascular growth.16,17
Materials and Methods The disc angiogenesis system (DAS) consists of a polyvinyl-alcohol foam disc 13 mm in diameter, covered on both flat sides by millipore filters and containing the test substance in a central pellet coated with a slow-release film.15 Vessels grow centripetally from the edge of subcutaneously implanted discs.15 Moderate vessel proliferation occurs spontaneously, and marked proliferation results from the use of angiogenic stimulants such as basic fibroblastic growth factor (bFGF). Therefore, the DAS is suited to measure the effects of both agonists and antagonists of angiogenesis.1 '17 In one series of experiments, sterile discs, containing the substances indicated in the Table (A), were implanted subcutaneously in the thorax of female BALB/c mice, 2 months old, using procedures described elsewhere.15,17 In a second series of experiments (Table, B) mice had, in addition to the angiogenesis disc, an osmotic minipump (Alzet #2002, 14 day; Alza, Palo Alto, Supported in part by Veterans Affairs Research Funds (FAJO04) and by Syntex Research. Accepted for publication January 3, 1992. Address reprint requests to Dr. Luis F. Fajardo, Laboratory Service (113), Veterans Affairs Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304.
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Table. TNF-a Angiogenesis Experiments A
Mice 10 10 10 10 10 10 10 10 15 11
B Mice
Disc
0.01 mrTNF 0.1 mrTNF 1.0 mrTNF 10.0 mrTNF 100.0 mrTNF mrTNF 200.0 1000.0 mrTNF 5000.0 mrTNF No cytokine (spontaneous growth control) bFGF 20 (positive control)
ng ng ng ng ng ng ng ng
6 6 6 6 6
Disc
Minipump mrTNF 5 ,ug mrTNF 10 ,ug
bFGF 20 ,ug
mrTNF 20 ,ug PBS 200 ,u PBS 200 ,ul
,ug
Two series of experiments are summarized in this table. A (left columns): Angiogenesis discs (containing, within a centrally located pellet, mouse rTNFa in increasing amounts) were placed subcutaneously in BALB/c mice. Positive controls had bFGF instead of TNF. Spontaneous growth controls had no cytokine. B (right columns): Mice had an angiogenesis disc and an osmotic minipump, both placed subcutaneously (diagram in Figure 2). The contents of each are listed. Positive controls had bFGF in the disc, no cytokine in the minipump. Spontaneous growth controls had no cytokines but contained phosphate-buffered 0.9% NaCI (PBS) in the minipump.
CA) placed also subcutaneously, through a different incision. Its delivering nipple was located 5 mm from the edge of the disc. The contents of disc and minipump are listed, for each group of mice, in the Table (B). Notice that TNF was placed only in the minipump. In all experiments, we used mouse recombinant TNFa (Glaxo, Geneva, Switzerland) kindly provided by Drs. P. Vassalli and G. Grau, Geneva; the specific activity was 3 x 107 U/mg. In each experiment [methyl-3H]-thymidine (Dupont Inc., specific activity = 6.7 Ci/mmole) (3HTdR) was injected intraperitoneally at 24, 18, and 12 hours before euthanasia (total dose, 200 ,uCi/mouse). A dye, Luconyl Blue, was injected intracardially 15 minutes before sacrifice to improve recognition of vessels for qualitative studies.15 Discs were extracted 14 days after implantation, fixed and embedded in paraffin. Fifteen, 6 ,um thick planar sections of each disc (including the entire circumference) were obtained: three for histologic study (H&E) and measurement of axial growth; one for measurement of total growth area; ten for scintillation counting; and one for cellular localization of 3HTdR by autoradiography. The growth area was measured by an automated, computer-assisted, digital, image analysis microscopic technique (IMAGE MEASURE/IP, version 4.0; Microscience, Inc., Federal Way, WA) using sections stained with toluidine blue.17 The total growth area, which in this system is consistently proportional to total vessel area, was converted from pixels to mm.2 Axial (radial) growth in the discs was measured in centimeters by projecting the sections at x1 00 magnification.15 In every disc of the second series of experiments (Table, B), the growth was separately measured along each of four equidistant radii: two radii in the one half of the disc proximal to the osmotic minipump and two radii in the one half of the disc distal to the pump. Proliferation, of all cells, was determined by 3HTdR
incorporation. Scintillation counting was performed after standard alkali solubilization and extraction of 10 adjacent paraffin sections from each disc. Identification and localization of the cells containing labeled thymidine was done by autoradiography (5 week exposure). Raw data (from measurements of axial growth, total area, 3HTdR incorporation, etc.) were analyzed in an IBM-PC/AT computer using the RSI statistical analysis software. Having obtained means, standard errors, and standard deviations for each group of discs, comparison of experimental and control groups were carried out using Student's t test.
Results The TNF placed in the center of the discs caused two separate and opposing effects. At concentrations of 0.01 to 1 ng/disc it stimulated angiogenesis, with a maximum effect at 0.1 ng (P < 0.01) for axial growth. As the dose of TNF increased, the angiogenic effect disappeared. Then a reverse effect, inhibition of angiogenesis, was observed at doses of 1 and 5 ,ug (P < 0.01). Figure 1 illustrates this dose effect as detected by axial (radial) measurements of growth. The same bimodal effect was evident by measurement of the total growth area (not shown). The inhibitory effect of high doses was observed also when the TNF was placed in minipumps, and delivered locally (subcutaneously) at the high rate of 15-60 ng/hr during 14 days (Figure 2). Pumps containing 20 ,ug of TNF produced a significant decrease in angiogenesis. Figure 2 shows also the level of spontaneous growth obtained in the (unstimulated) control discs, and the expectedly high angiogenic response to bFGF, in the positive control discs. The morphology of the fibrovascular growth observed in these experiments is illustrated in Figure 3. The type of growth observed in (a) and (c) was seen in
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0
a-i
z Q
F Iz I=
-
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w
0.01
0.1
1.0
10
100
1000
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Figure 1. Dose-deperdent bimodal effect of TNF on angiogenesis. All values have been normalized in relation to the control (spontaneous growth) values, wbich are indicated by a straight line at the 100% level. The solid line (left scale of percentages) indicates thefibrovascular growth as measured alongfour axes. The broken line (right scale ofpercentages) indicates the incorporation of tritiated thymidine as measured by scintillation counting. Error bars ± SEM. The TNF contained in the center of the discs is indicated in nanograms. Both fibrovascular growth and cell proliferation increased with the low doses of TNF and decreased with the high doses.
controls as well as in discs exposed to low (e.g., 1 ng) concentrations of TNF or to bFGF. The decrease in growth caused by increasing concentrations of TNF is illustrated in (b). Cellular proliferation, as indicated by 3HTdR incorporation, was enhanced by the low concentrations of TNF, and decreased by the high concentrations (Figure 1). Therefore the bimodal effect of TNF was not only applied to the total fibrovascular growth, but also to the proliferation of cells within such growth. Autoradiographs confirmed that thymidine incorporation occurred almost exclusively in endothelial cells and fibroblasts.
Our results are consistent on the one hand with those of Leibovitch et al,14 who observed angiogenesis in the rat cornea with 3.5 ng of mouse TNF, and on the other hand with those of BenEzra et al,19 who observed little or no angiogenesis in the rabbit cornea with much higher doses (50-1000 ng) of human TNF. The doses of TNF used in our minipump experiments (15-60 ng/hr) were within the range used by Piguet et al' for subcutaneous perfusion of TNF in the mouse (35-170 ng/hr). They observed proliferation of capillaries and other connective tissue constituents at low doses and necrosis at high doses.20 All these observations indicate that low doses of TNF are angiogenic. Two questions follow. First, by what mechanism does TNF exert angiogenic effects, and second, what is the relative importance of TNF and other mediators in angiogenesis? TNF is known to induce the production of prostaglandins by several cell types,6'8 and it has been suggested that production of prostaglandins by macrophages may mediate the angiogenic effect of TNF.19 We have summarized evidence that bFGF induces the production of prostaglandin E2 (PGE2) by microvascular endothelial cells, and that PGE2 augments the production of cyclic AMP in these cells thereby stimulating their proliferation.21 The angiogenic effects of bFGF and epidermal growth factor (EGF) in the disc angiogenesis system were abolished by systemic administration of drugs inhibiting prostaglandin synthesis.22 Thus prostaglandins are transducers of angiogenic signals by 20
E 15
0
-C 0
Discussion
O 10
The observations described above show that, using the quantitative disc angiogenesis assay, mouse recombinant TNF in low concentrations promotes, and in high concentrations inhibits angiogenesis. These findings explain, at least partially, discrepancies between previous in vitro11-13 and in vivo12,14 observations. Other factors that may have contributed to the discrepancies include variations in experimental designs and the use of TNF from one animal species in another. There are species differences in the sequence of amino acids in TNF7 and in receptor-ligand interactions,718 that could interfere with the biological effects of a heterologous TNF moiety. However, since murine TNF was found to induce angiogenesis in the chick chorioallantoic membrane,14 at least some responses to TNF cross wide species barriers.
c
'a x
0)5 n
Pump: 0 Disc: 0
0 FGF 20
TNF 5 0
TNF 10 0
TNF 20 0
Figure 2. Axial growth in discs exposed to the TNF releasedfrom adjacent, subcutaneous minipumps (as indicated in right upper diagram). Solid bars indicate growth in the one half of the disc proximal to the pump, and hatched bars in the one half distal to the pump. Legend below each set of bars indicates above the content of the minipump, and below the content of the disc, both in pg. 0 = no cytokine. Error bars = +SEM As expected, discs containing bFGF had significantly greater growth than control discs (fst set of bars). As in the previous eperiments (Figure 1) high doses of TNF inhibited fibrovascular growth (P < 0.01). In every group the growth was less in the side close to the mintpump (solid bars) than in the opposite side suggesting that the pump by itself-directly or indirectly-inhibited some growth.
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Figure 3. Peripheral growth area of discs in 6 p.m thick planar sections stained with hematoxylin and eosin. In each panel the bottom corresponds to theperiphery ofthe disc and the top is oriented toward the center of the disc. a (top left): Example of thefibrovascular growth seen in unstimulated (control) discs. Qualitatively similar growth occurred in discs stimulated by low doses (e.g., 1 ng) of TNF. Blood capillaries are identfied by the dye Luconyl Blue. Around capillaries there is a loose network offibroblasts, some leukocytes and occasional erythrocytes. The gray-pink irregular material is the matrix ofthe polyvinyl alcohol sponge, x 60. b (top rig ht): Reduced vascular growthlimited to the lower portion ofphotograph-in a disc containing 1 pg of TNF, X 60. c (bottom): Detail of growth in disc exposed to 20 pg of bFGF, showing branching capillaries, fibroblasts, leukocytes and erythrocytes, x200.
bFGF and EGF, and this may be also true for the angiogenic effect of TNF. Although the tissue concentration of this cytokine seems important, other aspects of TNF-induced angiogenesis are unclear. A recent immunohistochemical study of the cauterized, but otherwise untreated, mouse cornea did not demonstrate TNF in the infiltrating cells and capillary sprouts.23 The investigators concluded that the release of TNF by the infiltrating cells was of minor or no importance for the induction of sterile inflammatory angiogenesis in the cornea.23 To define the exact role of TNF in angiogenesis is an important goal since there is a growing number of pathologic conditions in which elevation of serum TNFa has been demonstrated.24-6 Also TNF is being promoted, and clinically tried, as an antineoplastic agent.8'27 Depending on the concentration of the exogenous cytokine reached in the neoplasm (and perineoplastic tissues) it may inhibit angiogenesis28 and therefore inhibit tumor growth, or it may stimulate angiogenesis and thus promote tumor growth. The poor antineoplastic performance of TNFa in human tumors may result partially from the latter.
Acknowledgment The authors thank Donna L. Buckley for assisting with manuscript preparation.
References 1. Folkman J: Tumor angiogenesis. Adv Cancer Res 1985,
43:175-203 2. Furcht LT: Critical factors controlling angiogenesis: cell products, cell matrix and growth factors. Lab Invest 1986, 55:505-509 3. Folkman J, Klagsbrun M: A family of angiogenic peptides. Nature 1987, 329:671-672 4. Madri JA, Williams SK, Wyatt T, Mezzio C: Capillary endothelial cell cultures: phenotypic modulation by matrix components. J Cell Biol 1983, 97:153-165 5. Madri JA, Kocher 0, Merwin JR, Bell L, Yannariello-Brown J: The interactions of vascular cells with solid phase (matrix) and soluble factors. J Cardiovasc Pharm 1989, 14(Suppl.
6):S70-S75
6. Beutler B, Cerami A: Cachectin: more than a tumor necrosis factor. N Engl J Med 1987, 316:379-385 7. Tracey KJ, Vlassara H, Cerami A: Cachectin/tumor necrosis factor. Lancet 1989, 1:1122-1125 8. Beutler B: The tumor necrosis factors: Cachectin and lymphotoxin. Hosp Pract 1990, 25:45-56 9. Jaattela M: Biologic activities and mechanism of action of TNF-o/cachectin. Lab Invest 1991, 64:724-742 10. Rosenbaum JT, Howes FL, Rubin RM, Samples JR: Ocular inflammatory effects of intravitreally injected tumor necrosis factor. Am J Pathol 1988, 133:47-53 11. Schweigerer L, Malerstein B, Gospodarowicz D: Tumor necrosis factor inhibits the proliferation of cultured capillary endothelial cells. Biophys Res Comm 1987,143:997-1004 12. Frater-Schr6der M, Risau W, Hallmann R, Gautschi P, Bohlen P: Tumor necrosis factor a, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc Natl Acad Sci 1987, 84:5277-5281 13. Sato N, Fukuda K, Nariuchi H, Sagara N: Tumor necrosis factor inhibiting angiogenesis in vitro. J Natl Cancer Inst 1987, 79:1383-1391 14. Leibovich SJ, Polverini PJ, Shepard HM, Wiseman DM, Shively V, Nuseir N: Macrophage-induced angiogenesis is mediated by tumor necrosis factor-a. Nature 1987, 329:630-632 15. Fajardo LF, Kowalski J, Kwan HH, Prionas SD, Allison AC: The disc angiogenesis system. Lab Invest 1988, 58:718724 16. Fajardo LF, Prionas SD, Kowalski J, Kwan HH: Hyperthermia inhibits angiogenesis. Radiat Res 1988, 114:297-306 17. Prionas SD, Kowalski J, Fajardo LF, Kaplan I, Kwan HH, Allison AC: Effects of x-irradiation on angiogenesis. Radiat
Res 1990,124:43-49 18. Ranges GE, Zlotnik A, Espervik T, Dinarello CA, Cerami A, Palladino MA: Tumor necrosis factor-alpha/cachectin is a growth factor for thymocytes: synergistic interaction with other cytokines. J Exp Med 1988, 167:1472-1478 19. BenEzra D, Hemo I, Maftzir G: In vivo angiogenic activity of interleukins. Arch Ophthalmol 1990, 108:573-576 20. Piguet PF, Grau GE, Vassalli P: Subcutaneous perfusion of tumor necrosis factor induces local proliferation of fibroblasts, capillaries, and epidermal cells, or massive tissue necrosis. Am J Pathol 1990, 136:103-1 10 21. Allison AC, Kowalski J: Prostaglandins as transducers of proliferation signals in microvascular endothelial cells and the pharmacological control of angiogenesis. Vascular Endothelium, Receptors and Transduction Mechanisms. Edited by J. D. Catravas, C. N. Gillis, and U. S. Ryan. New York, Plenum Press, 1989, pp 99-110 22. Kowalski J, Kwan HH, Prionas SD, Allison AC, Fajardo LF:
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Characterization of capillary growth in an in vivo angiogenesis model. Exp Mol Pathol 1992, 56:(in press) 23. Sunderkotter C, Roth J Sorg C: Immunohistochemical detection of bFGF and TNF-a in the course of inflammatory angiogenesis in the mouse cornea. Am J Pathol 1990, 137:511-515 24. Grau GE, Fajardo LF, Piguet PF, Allet B, Lambert PH, Vassalli P: Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 1987, 237:1210-1212 25. Girardin E, Grau GE, Dayer JM, Roux-Lombard P, the 15 Study Group, Lambert PH: Tumor necrosis factor and inter-
leukin-1 in the serum of children with severe infectious purpura. N Engl J Med 1988, 319:397-400 26. Fajardo LF, Grau GE: Tumor necrosis factor in human disease. West J Med 1991, 154:88-89 27. Creagan ET, Kovach JS, Moertel CG, Frytak S: A phase clinical trial of recombinant human tumor necrosis factor. Cancer 1988, 62:2467-2471 28. Srinivasan JM, Fajardo LF, Hahn GM: Mechanism of antitumor activity of tumor necrosis factor with hyperthermia in a tumor necrosis factor a-resistant tumor. J Natl Cancer Inst 1990, 82:1904-1910
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Rapid Communication Dual Role of Tumor Necrosis Factor-a in Angiogenesis Luis F. Fajardo,* Helen H. Kwan,* Joe Kowalski,t Stavros D. Prionas,t and Anthony C. Allisont From the Departments of Pathology' and Radiation Oncology, Stanford University School ofMedicine, and the Veterans Affairs Medical Center, Palo Alto, California, and the Institute ofBiological Science,t Syntex Research,
Palo Alto, California
The role of tumor necrosis factor-ot (TNF; cachectin) in angiogenesis has been controversial. In vitro TNF inhibits proliferation of endothelial cells (EC) whereas in the cornea it appears to stimulate vessel growth. The authors tested TNF in their recently developed disc angiogenesis system (DAS), designed to measure the proliferation of microvessels. The DAS, implanted subcutaneously in mice, was either fitted with a central pellet containing mouse recombinant TAF (mrTNF), or exposed to mrTNF delivered subcutaneously by an osmotic minipump. Low doses of mrTNF (0. 01-1 ng) induced angiogenesis, which was maximum at 0.1 ng whereas high doses (1, and 5 jig) inhibited it. Subcutaneous mrTNF delivered at the (high) rate of 15-60 ng/hr for 14 days inhibited angiogenesis. These results indicate bimodal; dosedependent opposing effects and explain some of the in vitro versus in vivo paradoxical results. TAF (native or exogenous) may have opposing effects on micovessels of neoplasms and inflammatory reactions, depending on its local tissue concentrations. (Am J Pathol 1992, 140:539-544)
Angiogenesis, the proliferation of microvessels in the developed vertebrate, is a process particularly important in neoplasia, inflammation, and wound repair.1'2 Some cell products2'3 and stromal components 4'5 that regulate it have been identified. Among the substances that may influence angiogenesis is tumor necrosis factor-a (TNF), a cytokine mainly produced by macrophages. TNF has
multiple well-recognized biological activities.6'9 However, its function in angiogenesis is incompletely understood because of discrepant observations. Several in vitro studies indicate that TNF inhibits the proliferation of cultured capillary endothelial cells (EC), 1l12 or microvascular sprouts,13 and is cytotoxic for stimulated EC.1" Those effects suggest that in vivo TNF should inhibit angiogenesis. However, two studies reported that in the cornea of rabbits and rats, human and mouse TNF, respectively, induced angiogenesis.12,14 To clarify this paradox, we have tested TNF in a new in vivo system (developed in our laboratory15) which allows easy quantification of vascular growth.16,17
Materials and Methods The disc angiogenesis system (DAS) consists of a polyvinyl-alcohol foam disc 13 mm in diameter, covered on both flat sides by millipore filters and containing the test substance in a central pellet coated with a slow-release film.15 Vessels grow centripetally from the edge of subcutaneously implanted discs.15 Moderate vessel proliferation occurs spontaneously, and marked proliferation results from the use of angiogenic stimulants such as basic fibroblastic growth factor (bFGF). Therefore, the DAS is suited to measure the effects of both agonists and antagonists of angiogenesis.1 '17 In one series of experiments, sterile discs, containing the substances indicated in the Table (A), were implanted subcutaneously in the thorax of female BALB/c mice, 2 months old, using procedures described elsewhere.15,17 In a second series of experiments (Table, B) mice had, in addition to the angiogenesis disc, an osmotic minipump (Alzet #2002, 14 day; Alza, Palo Alto, Supported in part by Veterans Affairs Research Funds (FAJO04) and by Syntex Research. Accepted for publication January 3, 1992. Address reprint requests to Dr. Luis F. Fajardo, Laboratory Service (113), Veterans Affairs Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304.
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Table. TNF-a Angiogenesis Experiments A
Mice 10 10 10 10 10 10 10 10 15 11
B Mice
Disc
0.01 mrTNF 0.1 mrTNF 1.0 mrTNF 10.0 mrTNF 100.0 mrTNF mrTNF 200.0 1000.0 mrTNF 5000.0 mrTNF No cytokine (spontaneous growth control) bFGF 20 (positive control)
ng ng ng ng ng ng ng ng
6 6 6 6 6
Disc
Minipump mrTNF 5 ,ug mrTNF 10 ,ug
bFGF 20 ,ug
mrTNF 20 ,ug PBS 200 ,u PBS 200 ,ul
,ug
Two series of experiments are summarized in this table. A (left columns): Angiogenesis discs (containing, within a centrally located pellet, mouse rTNFa in increasing amounts) were placed subcutaneously in BALB/c mice. Positive controls had bFGF instead of TNF. Spontaneous growth controls had no cytokine. B (right columns): Mice had an angiogenesis disc and an osmotic minipump, both placed subcutaneously (diagram in Figure 2). The contents of each are listed. Positive controls had bFGF in the disc, no cytokine in the minipump. Spontaneous growth controls had no cytokines but contained phosphate-buffered 0.9% NaCI (PBS) in the minipump.
CA) placed also subcutaneously, through a different incision. Its delivering nipple was located 5 mm from the edge of the disc. The contents of disc and minipump are listed, for each group of mice, in the Table (B). Notice that TNF was placed only in the minipump. In all experiments, we used mouse recombinant TNFa (Glaxo, Geneva, Switzerland) kindly provided by Drs. P. Vassalli and G. Grau, Geneva; the specific activity was 3 x 107 U/mg. In each experiment [methyl-3H]-thymidine (Dupont Inc., specific activity = 6.7 Ci/mmole) (3HTdR) was injected intraperitoneally at 24, 18, and 12 hours before euthanasia (total dose, 200 ,uCi/mouse). A dye, Luconyl Blue, was injected intracardially 15 minutes before sacrifice to improve recognition of vessels for qualitative studies.15 Discs were extracted 14 days after implantation, fixed and embedded in paraffin. Fifteen, 6 ,um thick planar sections of each disc (including the entire circumference) were obtained: three for histologic study (H&E) and measurement of axial growth; one for measurement of total growth area; ten for scintillation counting; and one for cellular localization of 3HTdR by autoradiography. The growth area was measured by an automated, computer-assisted, digital, image analysis microscopic technique (IMAGE MEASURE/IP, version 4.0; Microscience, Inc., Federal Way, WA) using sections stained with toluidine blue.17 The total growth area, which in this system is consistently proportional to total vessel area, was converted from pixels to mm.2 Axial (radial) growth in the discs was measured in centimeters by projecting the sections at x1 00 magnification.15 In every disc of the second series of experiments (Table, B), the growth was separately measured along each of four equidistant radii: two radii in the one half of the disc proximal to the osmotic minipump and two radii in the one half of the disc distal to the pump. Proliferation, of all cells, was determined by 3HTdR
incorporation. Scintillation counting was performed after standard alkali solubilization and extraction of 10 adjacent paraffin sections from each disc. Identification and localization of the cells containing labeled thymidine was done by autoradiography (5 week exposure). Raw data (from measurements of axial growth, total area, 3HTdR incorporation, etc.) were analyzed in an IBM-PC/AT computer using the RSI statistical analysis software. Having obtained means, standard errors, and standard deviations for each group of discs, comparison of experimental and control groups were carried out using Student's t test.
Results The TNF placed in the center of the discs caused two separate and opposing effects. At concentrations of 0.01 to 1 ng/disc it stimulated angiogenesis, with a maximum effect at 0.1 ng (P < 0.01) for axial growth. As the dose of TNF increased, the angiogenic effect disappeared. Then a reverse effect, inhibition of angiogenesis, was observed at doses of 1 and 5 ,ug (P < 0.01). Figure 1 illustrates this dose effect as detected by axial (radial) measurements of growth. The same bimodal effect was evident by measurement of the total growth area (not shown). The inhibitory effect of high doses was observed also when the TNF was placed in minipumps, and delivered locally (subcutaneously) at the high rate of 15-60 ng/hr during 14 days (Figure 2). Pumps containing 20 ,ug of TNF produced a significant decrease in angiogenesis. Figure 2 shows also the level of spontaneous growth obtained in the (unstimulated) control discs, and the expectedly high angiogenic response to bFGF, in the positive control discs. The morphology of the fibrovascular growth observed in these experiments is illustrated in Figure 3. The type of growth observed in (a) and (c) was seen in
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± I.
II
0
a-i
z Q
F Iz I=
-
0
w
0.01
0.1
1.0
10
100
1000
10,000
TNF (ng)
Figure 1. Dose-deperdent bimodal effect of TNF on angiogenesis. All values have been normalized in relation to the control (spontaneous growth) values, wbich are indicated by a straight line at the 100% level. The solid line (left scale of percentages) indicates thefibrovascular growth as measured alongfour axes. The broken line (right scale ofpercentages) indicates the incorporation of tritiated thymidine as measured by scintillation counting. Error bars ± SEM. The TNF contained in the center of the discs is indicated in nanograms. Both fibrovascular growth and cell proliferation increased with the low doses of TNF and decreased with the high doses.
controls as well as in discs exposed to low (e.g., 1 ng) concentrations of TNF or to bFGF. The decrease in growth caused by increasing concentrations of TNF is illustrated in (b). Cellular proliferation, as indicated by 3HTdR incorporation, was enhanced by the low concentrations of TNF, and decreased by the high concentrations (Figure 1). Therefore the bimodal effect of TNF was not only applied to the total fibrovascular growth, but also to the proliferation of cells within such growth. Autoradiographs confirmed that thymidine incorporation occurred almost exclusively in endothelial cells and fibroblasts.
Our results are consistent on the one hand with those of Leibovitch et al,14 who observed angiogenesis in the rat cornea with 3.5 ng of mouse TNF, and on the other hand with those of BenEzra et al,19 who observed little or no angiogenesis in the rabbit cornea with much higher doses (50-1000 ng) of human TNF. The doses of TNF used in our minipump experiments (15-60 ng/hr) were within the range used by Piguet et al' for subcutaneous perfusion of TNF in the mouse (35-170 ng/hr). They observed proliferation of capillaries and other connective tissue constituents at low doses and necrosis at high doses.20 All these observations indicate that low doses of TNF are angiogenic. Two questions follow. First, by what mechanism does TNF exert angiogenic effects, and second, what is the relative importance of TNF and other mediators in angiogenesis? TNF is known to induce the production of prostaglandins by several cell types,6'8 and it has been suggested that production of prostaglandins by macrophages may mediate the angiogenic effect of TNF.19 We have summarized evidence that bFGF induces the production of prostaglandin E2 (PGE2) by microvascular endothelial cells, and that PGE2 augments the production of cyclic AMP in these cells thereby stimulating their proliferation.21 The angiogenic effects of bFGF and epidermal growth factor (EGF) in the disc angiogenesis system were abolished by systemic administration of drugs inhibiting prostaglandin synthesis.22 Thus prostaglandins are transducers of angiogenic signals by 20
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The observations described above show that, using the quantitative disc angiogenesis assay, mouse recombinant TNF in low concentrations promotes, and in high concentrations inhibits angiogenesis. These findings explain, at least partially, discrepancies between previous in vitro11-13 and in vivo12,14 observations. Other factors that may have contributed to the discrepancies include variations in experimental designs and the use of TNF from one animal species in another. There are species differences in the sequence of amino acids in TNF7 and in receptor-ligand interactions,718 that could interfere with the biological effects of a heterologous TNF moiety. However, since murine TNF was found to induce angiogenesis in the chick chorioallantoic membrane,14 at least some responses to TNF cross wide species barriers.
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Figure 3. Peripheral growth area of discs in 6 p.m thick planar sections stained with hematoxylin and eosin. In each panel the bottom corresponds to theperiphery ofthe disc and the top is oriented toward the center of the disc. a (top left): Example of thefibrovascular growth seen in unstimulated (control) discs. Qualitatively similar growth occurred in discs stimulated by low doses (e.g., 1 ng) of TNF. Blood capillaries are identfied by the dye Luconyl Blue. Around capillaries there is a loose network offibroblasts, some leukocytes and occasional erythrocytes. The gray-pink irregular material is the matrix ofthe polyvinyl alcohol sponge, x 60. b (top rig ht): Reduced vascular growthlimited to the lower portion ofphotograph-in a disc containing 1 pg of TNF, X 60. c (bottom): Detail of growth in disc exposed to 20 pg of bFGF, showing branching capillaries, fibroblasts, leukocytes and erythrocytes, x200.
bFGF and EGF, and this may be also true for the angiogenic effect of TNF. Although the tissue concentration of this cytokine seems important, other aspects of TNF-induced angiogenesis are unclear. A recent immunohistochemical study of the cauterized, but otherwise untreated, mouse cornea did not demonstrate TNF in the infiltrating cells and capillary sprouts.23 The investigators concluded that the release of TNF by the infiltrating cells was of minor or no importance for the induction of sterile inflammatory angiogenesis in the cornea.23 To define the exact role of TNF in angiogenesis is an important goal since there is a growing number of pathologic conditions in which elevation of serum TNFa has been demonstrated.24-6 Also TNF is being promoted, and clinically tried, as an antineoplastic agent.8'27 Depending on the concentration of the exogenous cytokine reached in the neoplasm (and perineoplastic tissues) it may inhibit angiogenesis28 and therefore inhibit tumor growth, or it may stimulate angiogenesis and thus promote tumor growth. The poor antineoplastic performance of TNFa in human tumors may result partially from the latter.
Acknowledgment The authors thank Donna L. Buckley for assisting with manuscript preparation.
References 1. Folkman J: Tumor angiogenesis. Adv Cancer Res 1985,
43:175-203 2. Furcht LT: Critical factors controlling angiogenesis: cell products, cell matrix and growth factors. Lab Invest 1986, 55:505-509 3. Folkman J, Klagsbrun M: A family of angiogenic peptides. Nature 1987, 329:671-672 4. Madri JA, Williams SK, Wyatt T, Mezzio C: Capillary endothelial cell cultures: phenotypic modulation by matrix components. J Cell Biol 1983, 97:153-165 5. Madri JA, Kocher 0, Merwin JR, Bell L, Yannariello-Brown J: The interactions of vascular cells with solid phase (matrix) and soluble factors. J Cardiovasc Pharm 1989, 14(Suppl.
6):S70-S75
6. Beutler B, Cerami A: Cachectin: more than a tumor necrosis factor. N Engl J Med 1987, 316:379-385 7. Tracey KJ, Vlassara H, Cerami A: Cachectin/tumor necrosis factor. Lancet 1989, 1:1122-1125 8. Beutler B: The tumor necrosis factors: Cachectin and lymphotoxin. Hosp Pract 1990, 25:45-56 9. Jaattela M: Biologic activities and mechanism of action of TNF-o/cachectin. Lab Invest 1991, 64:724-742 10. Rosenbaum JT, Howes FL, Rubin RM, Samples JR: Ocular inflammatory effects of intravitreally injected tumor necrosis factor. Am J Pathol 1988, 133:47-53 11. Schweigerer L, Malerstein B, Gospodarowicz D: Tumor necrosis factor inhibits the proliferation of cultured capillary endothelial cells. Biophys Res Comm 1987,143:997-1004 12. Frater-Schr6der M, Risau W, Hallmann R, Gautschi P, Bohlen P: Tumor necrosis factor a, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc Natl Acad Sci 1987, 84:5277-5281 13. Sato N, Fukuda K, Nariuchi H, Sagara N: Tumor necrosis factor inhibiting angiogenesis in vitro. J Natl Cancer Inst 1987, 79:1383-1391 14. Leibovich SJ, Polverini PJ, Shepard HM, Wiseman DM, Shively V, Nuseir N: Macrophage-induced angiogenesis is mediated by tumor necrosis factor-a. Nature 1987, 329:630-632 15. Fajardo LF, Kowalski J, Kwan HH, Prionas SD, Allison AC: The disc angiogenesis system. Lab Invest 1988, 58:718724 16. Fajardo LF, Prionas SD, Kowalski J, Kwan HH: Hyperthermia inhibits angiogenesis. Radiat Res 1988, 114:297-306 17. Prionas SD, Kowalski J, Fajardo LF, Kaplan I, Kwan HH, Allison AC: Effects of x-irradiation on angiogenesis. Radiat
Res 1990,124:43-49 18. Ranges GE, Zlotnik A, Espervik T, Dinarello CA, Cerami A, Palladino MA: Tumor necrosis factor-alpha/cachectin is a growth factor for thymocytes: synergistic interaction with other cytokines. J Exp Med 1988, 167:1472-1478 19. BenEzra D, Hemo I, Maftzir G: In vivo angiogenic activity of interleukins. Arch Ophthalmol 1990, 108:573-576 20. Piguet PF, Grau GE, Vassalli P: Subcutaneous perfusion of tumor necrosis factor induces local proliferation of fibroblasts, capillaries, and epidermal cells, or massive tissue necrosis. Am J Pathol 1990, 136:103-1 10 21. Allison AC, Kowalski J: Prostaglandins as transducers of proliferation signals in microvascular endothelial cells and the pharmacological control of angiogenesis. Vascular Endothelium, Receptors and Transduction Mechanisms. Edited by J. D. Catravas, C. N. Gillis, and U. S. Ryan. New York, Plenum Press, 1989, pp 99-110 22. Kowalski J, Kwan HH, Prionas SD, Allison AC, Fajardo LF:
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Characterization of capillary growth in an in vivo angiogenesis model. Exp Mol Pathol 1992, 56:(in press) 23. Sunderkotter C, Roth J Sorg C: Immunohistochemical detection of bFGF and TNF-a in the course of inflammatory angiogenesis in the mouse cornea. Am J Pathol 1990, 137:511-515 24. Grau GE, Fajardo LF, Piguet PF, Allet B, Lambert PH, Vassalli P: Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 1987, 237:1210-1212 25. Girardin E, Grau GE, Dayer JM, Roux-Lombard P, the 15 Study Group, Lambert PH: Tumor necrosis factor and inter-
leukin-1 in the serum of children with severe infectious purpura. N Engl J Med 1988, 319:397-400 26. Fajardo LF, Grau GE: Tumor necrosis factor in human disease. West J Med 1991, 154:88-89 27. Creagan ET, Kovach JS, Moertel CG, Frytak S: A phase clinical trial of recombinant human tumor necrosis factor. Cancer 1988, 62:2467-2471 28. Srinivasan JM, Fajardo LF, Hahn GM: Mechanism of antitumor activity of tumor necrosis factor with hyperthermia in a tumor necrosis factor a-resistant tumor. J Natl Cancer Inst 1990, 82:1904-1910
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